title: DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses.
abstract: Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems.
authors.affiliation: Karlsruhe Institute of Technology, Campus North, Institute of Functional Interfaces (IFG), Microbiology of Natural and Technical Interfaces Division, P.O. Box 3640, 76021 Karlsruhe, Germany.
authors.forename: Jessica Varela
authors.initials: JV
authors.lastname: Villarreal
authors.forename: Christina
authors.initials: C
authors.lastname: Jungfer
authors.forename: Ursula
authors.initials: U
authors.lastname: Obst
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwartz
mesh_terms: D001419:Bacteria / Q000302:isolation & purification; D018441:Biofilms; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D060766:Drinking Water / Q000382:microbiology*; D019286:Endopeptidase K / Q000378:metabolism*; D050296:Microbial Viability; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D014871:Water Microbiology / Q000592:standards*
chemical_list: D004269:DNA, Bacterial; D060766:Drinking Water; D003850:Deoxyribonuclease I; D019286:Endopeptidase K
keywords: DNase; Drinking water biofilms; Live/dead discrimination; PCR based analyses; Reference bacteria